This invention relates to nucleic acid and amino acid sequences of a human PINCH protein homolog and to the use of these sequences in the diagnosis, treatment, and prevention of cancer and reproductive disorders.
LIM proteins are a family of proteins that share a common structural domain. The LIM motif is so named because it was first described in three proteins from Drosophila melanogaster designated L, I, and M. The LIM motif is a cysteine-rich region with a characteristic pattern: [C-X-X-C-X17xc2x11-H-X-X-C]-X-X-[C-X-X-C-X17xc2x11-C-X-X-C]. LIM motifs form two loop structures, and coordinate a zinc ion within each loop.
The LIM motif has been identified in a variety of proteins, including transcription factors, cytoskeletal proteins, and signaling molecules. LIM proteins are involved in cell fate determination, growth regulation, and oncogenesis. At least twenty-three members of the LIM family have been described, from nematodes to humans. Some LIM proteins consist of one, two, or three repeats of the LIM motif (LIM-only proteins). Others contain a LIM motif with a homeodomain (LIM-HD proteins) or a protein kinase domain (LIM-PK). LIM-PK inhibits the Ras oncogene-mediated differentiation of neural PC12 cells. LIM-HD proteins interact with DNA as well as bind to other proteins and are implicated in the control of differentiation of specific cell types. Studies in C. elegans demonstrated that LIM-HD proteins are involved in control of cell differentiation. Lin-11, a LIM-HD protein, controls the asymmetric cell divisions during vulval development, while Mec-3 is required for the differentiation of mechanosensory neurons. (Way, J. C. and Chalfie, M. (1988) Cell 54:5-16; and Freyd, G. et al (1990) Nature 344:876-879.)
The LIM-only proteins have not been shown to bind DNA, although the LIM structure is similar to the zinc finger, a well-characterized DNA-binding domain. LIM-only proteins include the rat cysteine-rich intestinal protein (CRIP), the human RBTN1 and RBTN2 proteins, and the chicken zyxin protein. (Higuchi, O. et al (1997) Oncogene 14:1819-1825; Sanchez-Garcia, I. and Rabbitts, T. H. (1994) Trends Genet. 10:315-320; and Dawid, I. B. et al (1995) C.R. Acad. Sci. III 318:295-306.) The genes for RBTN1 and RBTN2 are located on chromosome 11. Translocation mutations of chromosome 11 are associated with specific human T-cell acute leukemias. Transgenic expression of RBTN1 or RBTN2 produces leukemia and lymphoma in mice. (McGuire, E. A. et al (1992) Mol. Cell. Biol. 12:4186-4196; Fisch, P. et al (1992) Oncogene 7:2389-2397.)
A LIM-only protein known as PINCH protein (particularly interesting new Cys-His protein) was recently cloned from a human fetal liver library. PINCH protein contains five repeats of the LIM motif. Messenger RNA for PINCH protein is widely expressed, particularly in reproductive tissues, heart, and peripheral blood leukocytes. (Rearden, A. (1994) Biochem. Biophys. Res. Commun. 201: 1124-1131).
The discovery of a new human PINCH protein homolog and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cancer and reproductive disorders.
The invention features a substantially purified polypeptide, human PINCH protein homolog (PINCH-PH), comprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant of PINCH-PH having at least 90% amino acid identity to the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
Additionally, the invention provides a composition comprising a polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising a sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide which is complementary to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding PINCH-PH under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified PINCH-PH having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of PINCH-PH,
The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of PINCH-PH.
The invention also provides a method for detecting a polynucleotide encoding PINCH-PH in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding PINCH-PH in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.